The specific aim of this project is to develop a system for quantitative analysis of spermatogenesis in vitro. The system will be optimized for conditions which promote differentiation of the male germ line. Culture media will be formulated using a computer program (simplex optimization). The male germ line shows regulation of terminal differentiation, cell cycling, and meiosis modulated by hormones, growth factors, and cellular interactions. The advantages of this system are minimal requirements for tissue, use of invertebrates, and accurate quantification. The system will not only yield valuable basic information about spermatogenesis, but will be useful for identifying toxins or teratogens affecting germ line development and for product safety testing. Cultures consist of small amounts of gonadal tissue from the sea urchin. Most cells of the male germ line differ in either DNA content or size. Quantification will be made by flow cytometry, in which suspensions of cells or nuclei, irradiated by laser light, will be evaluated for DNA content, size, and internal complexity. The earliest cells of the lineage will be pulse-labelled with bromodeoxyuridine. Their differentiation will be revealed by the appearance of BrdU in successively more differentiated populations. Data will be expressed as: 1) fraction of all cells represented by each cell type, and 2) fraction of each cell type labelled with BrdU. Graphed vs. time in culture, these data will reveal both rate and extent of differentiation.